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Estradiol, a female sex hormone and the predominant form of estrogen, has diverse effects throughout the brain including in learning and memory. Estradiol modulates several types of learning that depend on the dorsomedial striatum (DMS), a subregion of the basal ganglia involved in goal-directed learning, cued action-selection, and motor skills. A cellular basis of learning is synaptic plasticity, and the presence of extranuclear estradiol receptors ERα, ERβ, and G-protein-coupled estrogen receptor (GPER) throughout the DMS suggests that estradiol may influence rapid cellular actions including those involved in plasticity. To test whether estradiol affects synaptic plasticity in the DMS, corticostriatal long-term potentiation (LTP) was induced using theta-burst stimulation (TBS) in ex vivo brain slices from intact male and female C57BL/6 mice. Extracellular field recordings showed that female mice in the diestrous stage of the estrous cycle exhibited LTP similar to male mice, while female mice in estrus did not exhibit LTP. Furthermore, antagonists of ERα or GPER rescued LTP in estrous females and agonists of ERα or GPER reduced LTP in diestrous females. In males, activating ERα but not GPER reduced LTP. These results uncover an inhibitory action of estradiol receptors on cellular learning in the DMS and suggest a cellular mechanism underlying the impairment in certain types of DMS-based learning observed in the presence of high estradiol. Because of the dorsal striatum's role in substance use disorders, these findings may provide a mechanism underlying an estradiol-mediated progression from goal-directed to habitual drug use., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Lewitus and Blackwell.)

To date, studies of development have mainly focused on the embryonic stage and a short time thereafter. There has been little research on the whole life of an individual from childhood to aging and death. For the first time, we used noninvasive urinary proteome technology to track changes in several important developmental time points in a group of rats, covering 10 time points from childhood, adolescence, young adulthood, middle adulthood, and near-death in old age. Similar to previous studies on puberty, proteins were detected and they are involved in sexual or reproductive maturation, mature spermatozoa in seminiferous tubules (first seen), gonadal hormones, decline of estradiol, brain growth, and central nervous system myelination, and our differential protein enrichment pathways also included reproductive system development, tube development, response to hormone, response to estradiol, brain development, and neuron development. Similar to previous studies in young adults, proteins were detected and they are involved in musculoskeletal maturity, peak bone mass, development of the immune system, and growth and physical development, and our differential proteins enrichment pathways also included skeletal system development, bone regeneration, system development, immune system processes, myeloid leukocyte differentiation, and developmental growth. Studies on aging-related changes in neurons and neurogenesis have been reported, and we also found relevant pathways in aged rats, such as regulation of neuronal synaptic plasticity and positive regulation of long-term neuronal synaptic plasticity. However, at all time points throughout life, there were many biological pathways revealed by differential urinary protein enrichment involving multiple organs, tissues, systems, etc. that have not been mentioned in existing studies. This study shows comprehensive and detailed changes in rat lifetime development through the urinary proteome, helping to fill the gap in development research. Moreover, it provides a new approach to monitoring changes in human health and diseases of aging using the urinary proteome., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Corticotropin-releasing hormone (CRH) neurons are the primary neural population controlling the hypothalamic-pituitary-adrenal (HPA) axis and the secretion of adrenal stress hormones. Previous work has demonstrated that stress hormone secretion can be regulated by circulating levels of estradiol. However, the effect of estradiol on CRH neuron excitability is less clear. Here, we show that chronic estradiol replacement following ovariectomy increases two types of potassium channel currents in CRH neurons: fast inactivating voltage-gated A-type K+ channel currents (IA) and non-inactivating M-type K+ channel currents (IM). Despite the increase in K+ currents following estradiol replacement, there was no overall change in CRH neuron spiking excitability assessed with either frequency-current curves or current ramps. Together, these data reveal a complex picture whereby ovariectomy and estradiol replacement differentially modulate distinct aspects of CRH neuron and HPA axis function., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Melatonin, an important regulator of mammalian reproduction, is mainly produced in the pineal gland, and granulosa cells (GCs), the main mammalian ovarian secretory cells, synthesize melatonin and express melatonin receptors (MRs) MT1 and MT2. However, studies on melatonin regulation in GCs are lacking in sheep. In this study, we explored the effects of β-estradiol (E2) on melatonin production and MR expression in GCs. We cultured sheep GCs to analyze the expression of the melatonin rate-limiting enzymes AANAT and HIOMT and the effects of E2 on AANAT, HIOMT, and MR expression and melatonin synthesis. To determine whether estrogen receptors (ERs) mediated E2 action on melatonin secretion and MR expression, we assessed ERA and ERB expression in GCs and observed whether ER antagonists counterbalanced the effects of E2. GCs expressed AANAT and HIOMT mRNA, indicating that they transformed exogenous serotonin into melatonin. E2 inhibited melatonin production by downregulating AANAT, HIOMT, and MRs. GCs expressed ERA and ERB; ERA/ERB inhibitors abolished E2-mediated inhibition of melatonin secretion and MR expression. PHTPP upregulated melatonin secretion and MT1 expression in E2-treated GCs, but did not significantly affect AANAT and MT2 expression. In conclusion, melatonin secretion in GCs was inhibited by E2 through an ERA- and ERB-mediated process., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Estradiol (E2) is a major hormone controlling women fertility, in particular folliculogenesis. This steroid, which is locally produced by granulosa cells (GC) within ovarian follicles, controls the development and selection of dominant preovulatory follicles. E2 effects rely on a complex set of nuclear and extra-nuclear signal transduction pathways principally triggered by its nuclear receptors, ERα and ERβ. These transcription factors are differentially expressed within follicles, with ERβ being the predominant ER in GC. Several ERβ splice isoforms have been identified and display specific structural features, which greatly complicates the nature of ERβ-mediated E2 signaling. This review aims at providing a concise overview of the main actions of E2 during follicular growth, maturation, and selection in human. It also describes the current understanding of the various roles of ERβ splice isoforms, especially their influence on cell fate. We finally discuss how E2 signaling deregulation could participate in two ovarian pathogeneses characterized by either a follicular arrest, as in polycystic ovary syndrome, or an excess of GC survival and proliferation, leading to granulosa cell tumors. This review emphasizes the need for further research to better understand the molecular basis of E2 signaling throughout folliculogenesis and to improve the efficiency of ovarian-related disease therapies.

Polycomb complexes have traditionally been prescribed roles as transcriptional repressors, though increasing evidence demonstrate they can also activate gene expression. However, the mechanisms underlying positive gene regulation mediated by Polycomb proteins are poorly understood. Here, we show that RING1B, a core component of Polycomb Repressive Complex 1, regulates enhancer-promoter interaction of the bona fide estrogen-activated GREB1 gene. Systematic characterization of RNA:DNA hybrid formation (R-loops), nascent transcription and RNA Pol II activity upon estrogen administration revealed a key role of RING1B in gene activation by regulating R-loop formation and RNA Pol II elongation. We also found that the estrogen receptor alpha (ERα) and RNA are both necessary for full RING1B recruitment to estrogen-activated genes. Notably, RING1B recruitment was mostly unaffected upon RNA Pol II depletion. Our findings delineate the functional interplay between RING1B, RNA and ERα to safeguard chromatin architecture perturbations required for estrogen-mediated gene regulation and highlight the crosstalk between steroid hormones and Polycomb proteins to regulate oncogenic programs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Estrogen hormones are implicated in a majority of breast cancers and estrogen receptor alpha (ER), the main nuclear factor mediating estrogen signaling, orchestrates a complex molecular circuitry that is not yet fully elucidated. Here, we investigated genome-wide DNA methylation, histone acetylation and transcription after estradiol (E2) deprivation and re-stimulation to better characterize the ability of ER to coordinate gene regulation. We found that E2 deprivation mostly resulted in DNA hypermethylation and histone deacetylation in enhancers. Transcriptome analysis revealed that E2 deprivation leads to a global down-regulation in gene expression, and more specifically of TET2 demethylase that may be involved in the DNA hypermethylation following short-term E2 deprivation. Further enrichment analysis of transcription factor (TF) binding and motif occurrence highlights the importance of ER connection mainly with two partner TF families, AP-1 and FOX. These interactions take place in the proximity of E2 deprivation-mediated differentially methylated and histone acetylated enhancers. Finally, while most deprivation-dependent epigenetic changes were reversed following E2 re-stimulation, DNA hypermethylation and H3K27 deacetylation at certain enhancers were partially retained. Overall, these results show that inactivation of ER mediates rapid and mostly reversible epigenetic changes at enhancers, and bring new insight into early events, which may ultimately lead to endocrine resistance., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Lung adenocarcinoma is the most common type of lung cancer in women. Our previous studies demonstrated that 17β-estradiol (E2) promoted lung adenocarcinoma cell proliferation and tumor growth through estrogen receptor ERα. Transcriptomic analysis suggested that E2 potentiated TNFα-NFκB signaling in ERα-expressing lung adenocarcinoma cells. This study further demonstrated that E2 increased TNFα receptor expression and TNFα-triggered NFκB activity in ERα-expressing cells. E2-activated ERα had no physical association with NFκB p65/p50 heterodimer but facilitated TNFα-initiated IκBα degradation, NFκB nuclear translocation, and S468/S536 phosphorylation of p65 essential for NFκB activity. While knockdown of ERα prevented E2 from boosting NFκB activity, antiestrogen ICI 182,780 stimulated NFκB activity like E2. Inhibition of GSK3β hampered E2:ERα-promoted NFκB activity and abolished S468 phosphorylation of p65, suggesting that GSK3β played a role in the E2-TNFα signaling crosstalk. In ERα-expressing cells, E2 and TNFα synergistically regulated many genes that were not typically responsive to either E2 or TNFα. Functional analysis of microarray data inferred that E2/TNFα-induced transcriptomic changes improved cell survival and movement. Viability and colony formation assays validated that E2 and TNFα together increased cisplatin tolerance of ERα-expressing cells. Wound healing assays also confirmed that E2/TNFα cotreatment increased cell migration in an ERα-dependent manner. E2/TNFα-induced dysregulation of genes such as cell survival and movement-associated genes, proto-oncogenes, metallothioneins and histone core genes was correlated with poor overall survival in patients. In summary, E2 and TNFα engaged in an ERα-dependent positive crosstalk in lung adenocarcinoma cells, consequently increasing NFκB activation, cisplatin tolerance and cell migration and worsening prognosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Oxygen causes white matter damage in preterm infants and male sex is a major risk factor for poor neurological outcome, which speculates the role of steroid hormones in sex-based differences. Preterm birth is accompanied by a drop in 17β-estradiol (E2) and progesterone along with increased levels of fetal zone steroids (FZS). We performed a sex-based analysis on the FZS concentration differences in urine samples collected from preterm and term infants. We show that, in preterm urine samples, the total concentration of FZS, and in particular the 16α-OH-DHEA concentration, is significantly higher in ill female infants as compared to males. Since we previously identified Nup133 as a novel target protein affected by hyperoxia, here we studied the effect of FZS, allopregnanolone (Allo) and E2 on differentiation and Nup133 signaling using mouse-derived primary oligodendrocyte progenitor cells (OPCs). We show that the steroids could reverse the effect of hyperoxia-mediated downregulation of Nup133 in cultured male OPCs. The addition of FZS and E2 protected cells from oxidative stress. However, E2, in presence of 16α-OH-DHEA, showed a negative effect on male cells. These results assert the importance of sex-based differences and their potential implications in preterm stress response.

The corpus luteum (CL) is a temporary endocrine gland vital for pregnancy establishment and maintenance. Estradiol-17β (E2) is the major embryonic signal in pigs supporting the CL's function. The mechanisms of the luteoprotective action of E2 are still unclear. The present study aimed to determine the effect of E2 on luteal expression of factors involved in CL function. An in vivo model of intrauterine E2 infusions was applied. Gilts on day 12 of pregnancy and the estrous cycle were used as referential groups. Concentrations of E2 and progesterone were elevated in CLs of gilts receiving E2 infusions, compared to placebo-treated gilts. Estradiol-17β stimulated luteal expression of DNA-methyltransferase 1 (DNMT1), but decreased expression of DNMT3B gene and protein, as well as DNMT3A protein. Similar results for DNMT3A and 3B were observed in CLs on day 12 of pregnancy compared to day 12 of the estrous cycle. Intrauterine infusions of E2 altered luteal expression of the genes involved in CL function: PTGFR , PTGES , STAR , HSD17B1 , CYP19A1, and PGRMC1 . Our findings indicate a role for E2 in expression regulation of factors related to CL function and a novel potential for E2 to regulate DNA methylation as putative physiological mechanisms controlling luteal gene expression.

Lactobacilli and estrogens play essential roles in vaginal homeostasis. We investigated the potential direct effect of 17β-estradiol on a vaginal strain of Lactobacillus crispatus, the major bacterial species of the vaginal microbiota. 17β-estradiol (10 -6 to 10 -10  M) had no effect on L. crispatus growth, but markedly affected the membrane dynamics of this bacterium. This effect appeared consistent with a signal transduction process. The surface polarity and aggregation potential of the bacterium were unaffected by exposure to 17β-estradiol, but its mean size was significantly reduced. 17β-estradiol also promoted biosurfactant production by L. crispatus and adhesion to vaginal VK2/E6E7 cells, but had little effect on bacterial biofilm formation activity. Bioinformatic analysis of L. crispatus identified a membrane lipid raft-associated stomatin/prohibitin/flotillin/HflK domain containing protein as a potential 17β-estradiol binding site. Overall, our results reveal direct effects of 17β-estradiol on L. crispatus. These effects are of potential importance in the physiology of the vaginal environment, through the promotion of lactobacillus adhesion to the mucosa and protection against pathogens.

The roles of sex and sex-hormones on the metabolic consequences of intermittent hypoxia (IH, a reliable model of sleep apnea) are unknown. We used intact male or female mice and ovariectomized (OVX) females treated with vehicle (Veh) or estradiol (E 2 ) and exposed to normoxia (Nx) or IH (6% O 2 , 10 cycles/h, 12 h/day, 2 wk). Mice were then fasted for 6 h, and we measured fasting glucose and insulin levels and performed insulin or glucose tolerance tests (ITT or GTT). We also assessed liver concentrations of glycogen, triglycerides (TGs), and expression levels of genes involved in aerobic or anaerobic metabolism. In males, IH lowered fasting levels of glucose and insulin, slightly improved glucose tolerance, but altered glucose tolerance in females. In OVX-Veh females, IH reduced fasting glucose and insulin levels and strongly impaired glucose tolerance. E 2 supplementation reversed these effects and improved homeostasis model assessment of β-cell function (HOMA-β), a marker of pancreatic glucose-induced insulin released. IH decreased liver TG concentration in males and slightly increased glycogen in OVX-Veh females. Liver expression of glycolytic ( Ldha ) and mitochondrial (citrate synthase, Pdha1 ) genes was reduced by IH in males and in OVX-Veh females, but not in intact or OVX-E 2 females. We conclude that 1 ) IH reduced fasting levels of glycemia in males and in ovariectomized females. 2 ) IH improves glucose tolerance only in males. 3 ) In females IH decreased glucose tolerance, this effect was amplified by ovariectomy, and reversed by E 2 supplementation. 4 ) During IH exposures, E 2 supplementation appears to improve pancreatic β cells functions. NEW & NOTEWORTHY We assessed fasting glycemic control, and tolerance to insulin and glucose in male and female mice exposed to intermittent hypoxia. IH improves glucose tolerance in males but had opposite effects in females. This response was amplified following ovariectomy in females and prevented by estradiol supplementation. Metabolic consequences of IH differ between males and females and are regulated by estradiol in female mice.

Animal taxa show remarkable variability in sexual reproduction, where separate sexes, or gonochorism, is thought to have evolved from hermaphroditism for most cases. Hermaphroditism accounts for 5% in animals, and sequential hermaphroditism has been found in teleost. In this study, we characterized a novel form of the transient hermaphroditic stage in little yellow croaker ( Larimichthys polyactis ) during early gonadal development. The ovary and testis were indistinguishable from 7 to 40 days post-hatching (dph). Morphological and histological examinations revealed an intersex stage of male gonads between 43 and 80 dph, which consist of germ cells, somatic cells, efferent duct, and early primary oocytes (EPOs). These EPOs in testis degenerate completely by 90 dph through apoptosis yet can be rescued by exogenous 17- β -estradiol. Male germ cells enter the mitotic flourishing stage before meiosis is initiated at 180 dph, and they undergo normal spermatogenesis to produce functional sperms. This transient hermaphroditic stage is male-specific, and the ovary development appears to be normal in females. This developmental pattern is not found in the sister species Larimichthys crocea or any other closely related species. Further examinations of serum hormone levels indicate that the absence of 11-ketotestosterone and elevated levels of 17- β -estradiol delineate the male intersex gonad stage, providing mechanistic insights on this unique phenomenon. Our research is the first report on male-specific transient hermaphroditism and will advance the current understanding of fish reproductive biology. This unique gonadal development pattern can serve as a useful model for studying the evolutionary relationship between hermaphroditism and gonochorism, as well as teleost sex determination and differentiation strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xie, Li, Zhan, Liu, Tan, Wang and Lou.)

While human vaginal pH in childbearing-age women is conclusively acidic, the mouse vaginal pH is reported as being near neutral. However, this information appears to be somewhat anecdotal with respect to vulvovaginal candidiasis, as such claims in the literature frequently lack citations of studies that specifically address this physiological factor. Given the disparate pH between mice and humans, the role of exogenous hormones and colonization by the fungal pathogen Candida albicans in shaping vaginal pH was assessed. Use of a convenient modified vaginal lavage technique with the pH indicator dye phenol red demonstrated that indeed vaginal pH was near neutral (7.2 ± 0.24) and was not altered by delivery of progesterone or estrogen in C57BL/6 mice. These trends were conserved in DBA/2 and CD-1 mouse backgrounds, commonly used in the mouse model of vaginitis. It was also determined that vaginal colonization with C. albicans did not alter the globally neutral vaginal pH over the course of one week. Construction and validation of a C. albicans reporter strain expressing GFPy, driven by the pH-responsive PHR1 promoter, confirmed the murine vaginal pH to be at least ≥6.0. Collectively, our data convincingly demonstrate a stable and conserved near neutrality of the mouse vaginal pH during vulvovaginal candidiasis and should serve as a definitive source for future reference. Implications and rationale for disparate pH in this model system are also discussed., (Copyright © 2021 American Society for Microbiology.)

Genomic sequence and gene expression association studies in animals and humans have identified genes that may be integral in the pathogenesis of various diseases. CD14 (cluster of differentiation 14)-a cell surface protein involved in innate immune system activation-is one such gene associated with cardiovascular and hypertensive disease. We previously showed that this gene is upregulated in renal macrophages of Dahl salt-sensitive animals fed a high-salt diet; here we test the hypothesis that CD14 contributes to the elevated pressure and renal injury observed in salt-sensitive hypertension. Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), we created a targeted mutation in the CD14 gene on the Dahl SS (SS/JrHSDMcwi) background and validated the absence of CD14 peptides via mass spectrometry. Radiotelemetry was used to monitor blood pressure in wild-type and CD14 -/ - animals challenged with high salt and identified infiltrating renal immune cells via flow cytometry. Germline knockout of CD14 exacerbated salt-sensitive hypertension and renal injury in female animals but not males. CD14 -/- females demonstrated increased infiltrating macrophages but no difference in infiltrating lymphocytes. Transplant of CD14 +/+ or CD14 -/- bone marrow was used to isolate the effects of CD14 knockout to hematopoietic cells and confirmed that the differential phenotype observed was due to knockout of CD14 in hematopoietic cells. Ovariectomy was used to remove the influence of female sex hormones, which completely abrogated the effect of CD14 knockout. These studies provide a novel treatment target and evidence of a new dichotomy in immune activation between sexes within the context of hypertensive disease where CD14 regulates immune cell activation and renal injury.

Metabolic dysfunction underlies several chronic diseases, many of which are exacerbated by obesity. Dietary interventions can reverse metabolic declines and slow aging, although compliance issues remain paramount. 17α-estradiol treatment improves metabolic parameters and slows aging in male mice. The mechanisms by which 17α-estradiol elicits these benefits remain unresolved. Herein, we show that 17α-estradiol elicits similar genomic binding and transcriptional activation through estrogen receptor α (ERα) to that of 17β-estradiol. In addition, we show that the ablation of ERα completely attenuates the beneficial metabolic effects of 17α-E2 in male mice. Our findings suggest that 17α-E2 may act through the liver and hypothalamus to improve metabolic parameters in male mice. Lastly, we also determined that 17α-E2 improves metabolic parameters in male rats, thereby proving that the beneficial effects of 17α-E2 are not limited to mice. Collectively, these studies suggest ERα may be a drug target for mitigating chronic diseases in male mammals., Competing Interests: SM, NH, MN, AR, RS, SA, MA, AU, MS, JH, DH, WF, MS No competing interests declared, (© 2020, Mann et al.)

Objective: Estrogen protects animals from obesity through estrogen receptor α (ERα), partially by inhibiting overeating in animals fed ad libitum. However, the effects of estrogen on feeding behavior in hungry animals remain unclear. In this study, we examined the roles of 17β-estradiol (E2) and ERα in the regulation of feeding in hungry female animals and explored the underlying mechanisms., Methods: Wild-type female mice with surgical depletion of endogenous estrogens were used to examine the effects of E2 supplementation on acute refeeding behavior after starvation. ERα-C451A mutant mice deficient in membrane-bound ERα activity and ERα-AF2 0 mutant mice lacking ERα transcriptional activity were used to further examine mechanisms underlying acute feeding triggered by either fasting or central glucopenia (induced by intracerebroventricular injections of 2-deoxy-D-glucose). We also used electrophysiology to explore the impact of these ERα mutations on the neural activities of ERα neurons in the hypothalamus., Results: In the wild-type female mice, ovariectomy reduced fasting-induced refeeding, which was restored by E2 supplementation. The ERα-C451A mutation, but not the ERα-AF2 0 mutation, attenuated acute feeding induced by either fasting or central glucopenia. The ERα-C451A mutation consistently impaired the neural responses of hypothalamic ERα neurons to hypoglycemia., Conclusion: In addition to previous evidence that estrogen reduces deviations in energy balance by inhibiting eating at a satiated state, our findings demonstrate the unexpected role of E2 that promotes eating in hungry mice, also contributing to the stability of energy homeostasis. This latter effect specifically requires membrane-bound ERα activity., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Ventromedial hypothalamic nucleus (VMN) gluco-regulatory transmission is subject to sex-specific control by estradiol. The VMN is characterized by high levels of aromatase expression., Methods: The aromatase inhibitor letrozole (LZ) was used with high-resolution microdissection/Western blot techniques to address the hypothesis that neuroestradiol exerts sex-dimorphic control of VMN neuronal nitric oxide synthase (nNOS) and glutamate decarboxylase 65/67 (GAD) protein expression. Glycogen metabolism impacts VMN nNOS and GAD profiles; here, LZ treatment effects on VMN glycogen synthase (GS) and phosphorylase brain- (GPbb; glucoprivic-sensitive) and muscle (GPmm; norepinephrine-sensitive) variant proteins were examined., Results: VMN aromatase protein content was similar between sexes. Intracerebroventricular LZ infusion of testes-intact male and ovariectomized, estradiol-replaced female rats blocked insulin-induced hypoglycemic (IIH) up-regulation of this profile. LZ exerted sex-contingent effects on basal VMN nNOS and GAD expression, but blocked IIH-induced NO stimulation and GAD suppression in each sex. Sex-contingent LZ effects on basal and hypoglycemic patterns of GPbb and GPmm expression occurred at distinctive levels of the VMN. LZ correspondingly down- or up-regulated baseline pyruvate recycling pathway marker protein expression in males (glutaminase) and females (malic enzyme-1), and altered INS effects on those proteins., Conclusions: Results infer that neuroestradiol is required in each sex for optimal VMN metabolic transmitter signaling of hypoglycemic energy deficiency. Sex differences in VMN GP variant protein levels and sensitivity to aromatase may correlate with sex-dimorphic glycogen mobilization during this metabolic stress. Neuroestradiol may also exert sex-specific effects on glucogenic amino acid energy yield by actions on distinctive enzyme targets in each sex.

Gender and menopause influence the severity and development manner of nonalcoholic steatohepatitis (NASH). Male p62/Sqstm1 and nuclear factor E2-related factor-2 (p62 and Nrf2) double-knockout (DKO) mice exhibit severe steatohepatitis caused by hyperphagia-induced obesity, overload of lipopolysaccharide (LPS) into the liver, and potentiation of the inflammatory response in Kupffer cells. However, the pathogenetic phenotype of steatohepatitis in female DKO mice remains unknown. Phenotypic changes of steatohepatitis in DKO mice were compared in terms of gender differences. Compared with DKO male mice, DKO female mice exhibited later onset of steatohepatitis with obesity after 30 weeks of age, as well as milder severity of hepatic inflammation and fibrosis. Serum estradiol was higher in female than male mice, with levels increasing up to 30 weeks of age before decreasing until 50 weeks of age (corresponding to the post-menopausal period). Fecal and serum LPS were lower in female mice than male mice, and inflammatory signaling in the liver was attenuated in female compared with male mice. Correlating with LPS levels, the composition of intestinal microbiota in female mice was different from male mice. Gender differences were observed for the development of steatohepatitis in DKO mice. Low-grade inflammatory hit in the liver under in vivo conditions of high estradiol may be attributable to the milder pathological features of steatohepatitis in female mice.

The estrogen membrane receptor GPR30 (also known as G-protein coupled receptor 30) has recently been shown to be involved in the regulation of oocyte maturation and cumulus expansion. However, whether GPR30 expression is regulated by gonadotropin stimulation and how it participates in the regulation of the maturation process is still not clear. In this study, we explored the mechanism underlying the synergy between luteinizing hormone and 17β-estradiol (17β-E 2 ) to improve the epidermal growth factor (EGF) response in cumulus oocyte complexes (COCs) during oocyte maturation in mice. The expression and distribution of GPR30, EGFR, and EGF-like growth factors were examined by real-time quantitative PCR, western blot, and immunofluorescence staining. Lyso-Tracker Red labeling was performed to detect the lysosomal activity in follicle granular cells (FGCs). Cumulus expansion of COCs was evaluated after in vitro maturation for 16 h. We found that EGF-like growth factors transmit LH signals to increase GRP30 levels by inhibiting protein degradation in lysosomes. Meanwhile, 17β-E 2 stimulates the GPR30 signaling pathway to increase EGF receptor levels, enhancing the response ability of EGF signaling in COCs and thus promoting cumulus expansion. In conclusion, our study reveals the synergistic mechanism between LH and estrogen in the regulation of cumulus expansion during oocyte maturation process.

Although hormones such as glucocorticoids have been broadly accepted in recent decades as general neuromodulators of memory processes, sex steroid hormones such as the potent oestrogen 17β-oestradiol have been less well recognized by the scientific community in this capacity. The predominance of females in studies of oestradiol and memory and the general (but erroneous) perception that oestrogens are 'female' hormones have probably prevented oestradiol from being more widely considered as a key memory modulator in both sexes. Indeed, although considerable evidence supports a crucial role for oestradiol in regulating learning and memory in females, a growing body of literature indicates a similar role in males. This Review discusses the mechanisms of oestradiol signalling and provides an overview of the effects of oestradiol on spatial, object recognition, social and fear memories. Although the primary focus is on data collected in females, effects of oestradiol on memory in males will be discussed, as will sex differences in the molecular mechanisms that regulate oestrogenic modulation of memory, which may have important implications for the development of future cognitive therapeutics.

The Fenton reaction (Fe 2+ +H 2 O 2 →Fe 3+ + • OH+OH - ) results in strong oxidative damage to macromolecules when iron (Fe) or hydrogen peroxide (H 2 O 2 ) are in excess. This study aims at comparing Fe 2+ +H 2 O 2 -induced oxidative damage to membrane lipids (lipid peroxidation, LPO) and protective effects of 17β-estradiol (a potential antioxidant) in porcine ovary and thyroid homogenates. Iron, as one of the Fenton reaction substrates, was used in the highest achievable concentrations. Thyroid or ovary homogenates were incubated in the presence of: (1st) FeSO 4 +H 2 O 2 with/without 17β-estradiol (1 mM; 100, 10.0, 1.0 µM; 100, 10.0, 1.0 nM; 100, 10.0, 1.0 pM); five experiments were performed with different FeSO 4 concentrations (2400, 1200, 600, 300, 150 µM); (2nd) FeSO 4 (2400, 1200, 600, 300, 150 µM)+H 2 O 2 with/without 17β-estradiol; three experiments were performed with three highest 17β-estradiol concentrations; (3rd) FeSO 4 (2400, 1200, 1100, 1000, 900, 800, 700, 600, 300, 150, 75 µM)+H 2 O 2 (5 mM). LPO level [MDA+4-HDA/mg protein] was measured spectrophotometrically. The basal LPO level is lower in ovary than in thyroid homogenates. However, experimentally-induced LPO was higher in the former tissue, which was confirmed for the three highest Fe 2+ concentrations (2400, 1200, 1100 µM). Exogenous 17β-estradiol (1 mM, 100, and 10 µM) reduced experimentally-induced LPO independently of iron concentration and that protective effect did not differ between tissues. The ovary, compared to the thyroid, reveals higher sensitivity to prooxidative effects of iron, however, it showed similar responsivity to protective 17β-estradiol activity. The therapeutic effect of 17β-estradiol against iron overload consequences should be considered with relation to both tissues.

The behavioral activation system (BAS) and the behavioral inhibition system (BIS) have been proposed to relate to stable traits that predict inter-individual differences in motivation. Prior reports point dopamine (DA) pathways, mainly including ventral tegmental area (VTA) and substantia nigra (SN), implicate in subserving reward-related functions associated with BAS and inhibitory functions related with BIS. However, as an important factor that affects DA releasing, it remains an open question whether the ovarian hormones may also be related to BIS/BAS. Here, to investigate effects of the estradiol (E2) and progesterone (PROG) on BIS/BAS and related DA pathways, we employed a BIS/BAS scale and the resting-state functional magnetic resonance imaging (fMRI) during the late follicular phase (FP) and the mid-luteal phase (LP). On the behavioral level, when women had high PROG levels, their E2 levels were found positively correlated with BIS scores, but those women whose PROG levels were low, their E2 levels were negative correlation with BIS scores. On the neural level, we demonstrated BAS was related with the VTA pathway, included brain reward regions of nucleus accumbens (NAc) and orbitofrontal cortex (OFC). Meanwhile, the BIS was correlated with the SN-dorsolateral prefrontal cortex (dlPFC) pathway. ROI-based resting-state functional connectivity (RSFC) analyses further revealed that, RSFC between the SN and dlPFC was modulated by ovarian hormones. With higher PROG levels, increased E2 levels among women were accompanied by stronger RSFC of the SN-dlPFC, but when PROG levels were low, E2 levels were negatively correlated with the SN-dlPFC RSFC. These findings revealed a combined enhancement effect of E2 and PROG on BIS, and the SN-dlPFC pathway was mainly involved in this process., Competing Interests: No authors have competing interests.

Naturally occurring cyclical changes in sex steroid hormones such as 17β-estradiol and progesterone can modulate neuron function and behavior in female mammals. One example is the estrous cycle in rats, which is composed of multiple phases. We previously reported evidence of differences between estrous cycle phases in excitatory synapse and intrinsic electrophysiological properties of rat nucleus accumbens core (AcbC) medium spiny neurons (MSNs). The AcbC is a nexus between the limbic and premotor systems and is integral for controlling motivated and reward-associated behaviors and disorders, which are sensitive to the estrous cycle and hormones. The present study expands our prior findings by testing whether circulating levels of estradiol and progesterone correlate with changes in MSN electrophysiology across estrous cycle phases. As part of this project, the excitatory synapse and intrinsic excitability properties of MSNs in late proestrus of adult female rats were assessed. Circulating levels of estradiol correlate with resting membrane potential, the time constant of the membrane, and rheobase. Circulating levels of progesterone correlate with miniature excitatory postsynaptic current (mEPSC) frequency and amplitude. Circulating levels of estradiol and progesterone together correlate with mEPSC amplitude, resting membrane potential, and input resistance. The late proestrus phase features a prominent and unique decrease in mEPSC frequency. These data indicate that circulating levels of estradiol and progesterone alone or in combination interact with specific MSN electrophysiological properties, indicating differential and synergistic roles of these hormones. Broadly, these findings illustrate the underlying endocrine actions regarding how the estrous cycle modulates MSN electrophysiology. NEW & NOTEWORTHY This research indicates that estradiol and progesterone act both differentially and synergistically to modulate neuron physiology in the nucleus accumbens core. These actions by specific hormones provide key data indicating the endocrine mechanisms underlying how the estrous cycle modulates neuron physiology in this region. Overall, these data reinforce that hormones are an important influence on neural physiology.

The menstrual cycle in humans and its analogous cycle in rodents, the estrous cycle, modulate brain function and behavior. Both cycles are characterized by the cyclical fluctuation of ovarian hormones including estrogens such as estradiol. Estradiol induces cycle- and sex-dependent differences in the phenotype and incidence of many behaviors, including those related to reward and motivation. The nucleus accumbens core (AcbC), a limbic and premotor system nexus region, directly regulates these behaviors. We previously showed that the estrous cycle modulates intrinsic excitability and excitatory synapse properties of medium spiny neurons (MSNs) in the AcbC. The identity of the underlying hormone mechanism is unknown, with estradiol being a prime candidate. The present study tests the hypothesis that estradiol induces estrous cycle-relevant differences in MSN electrophysiology. To accomplish this goal, a time- and dose-dependent estradiol replacement paradigm designed to simulate the rise of circulating estradiol levels across the estrous cycle was employed in ovariectomized adult female rats as well as a vehicle control group. Estradiol replacement decreased MSN excitability by modulating properties such as resting membrane potential, input resistance in both the linear and rectified ranges, and rheobase compared with vehicle-treated females. These differences in MSN excitability mimic those previously described regarding estrous cycle effects on MSN electrophysiology. Excitatory synapse properties were not modulated in response to this estradiol replacement paradigm. These data are the first to demonstrate that an estrous cycle-relevant estradiol exposure modulates MSN electrophysiology, providing evidence of the fundamental neuroendocrine mechanisms regulating the AcbC. NEW & NOTEWORTHY The present study shows, for the first time, that an estrous cycle-relevant estradiol exposure modulates nucleus accumbens neuron excitability. This evidence provides insight into the neuroendocrine mechanisms by which estradiol cyclically alters neuron properties during the estrous cycle. Overall, these data emphasize the significant influence of hormone action in the brain and especially individual neuron physiology.

Hypothalamic neurons show sex differences in neuritogenesis, female neurons have longer axons and higher levels of the neuritogenic factor neurogenin 3 (Ngn3) than male neurons in vitro. Moreover, the effect of 17-β-estradiol (E2) on axonal growth and Ngn3 expression is only found in male-derived neurons. To investigate whether sex chromosomes regulate these early sex differences in neuritogenesis by regulating the E2 effect on Ngn3, we evaluated the growth and differentiation of hypothalamic neurons derived from the "four core genotypes" mouse model, in which the factors of "gonadal sex" and "sex chromosome complement" are dissociated. We showed that sex differences in neurite outgrowth are determined by sex chromosome complement (XX > XY). Moreover, E2 increased the mRNA expression of Ngn3 and axonal length only in XY neurons. ERα/β expressions are regulated by sex chromosome complement; however, E2-effect on Ngn3 expression in XY neurons was only fully reproduced by PPT, a specific ligand of ERα, and prevented by MPP, a specific antagonist of ERα. Together our data indicate that sex chromosomes regulate early development of hypothalamic neurons by orchestrating not only sex differences in neuritogenesis, but also regulating the effect of E2 on Ngn3 expression through activation of ERα in hypothalamic neurons.

The study investigated the effect of normal and supraphysiological (resulting from gonadotropin-dependent ovarian stimulation) levels of estradiol (E2) and progesterone (P4) on mouse uterine aquaporin gene/protein (Aqp/AQP) expression on Day 1 (D1) and D4 of pregnancy. The study also examined the effect of ovarian stimulation on uterine luminal closure and uterine receptivity on D4 of pregnancy and embryo implantation on D5 and D7 of pregnancy. These analyses revealed that the expression of Aqp3, Aqp4, Aqp5 and Aqp8 is induced by E2 while the expression of Aqp1 and Aqp11 is induced by P4. Additionally, P4 inhibits E2 induction of Aqp3 and Aqp4 expression while E2 inhibits Aqp1 and Aqp11 expression. Aqp9, however, is constitutively expressed. Ovarian stimulation disrupts Aqp3, Aqp5 and Aqp8 expression on D4 and AQP1, AQP3 and AQP5 spatial expression on both D1 and D4, strikingly so in the myometrium. Interestingly, while ovarian stimulation has no overt effect on luminal closure and uterine receptivity, it reduces implantation events, likely through a disruption in myometrial activity and embryo development. The wider implication of this study is that ovarian stimulation, which results in supraphysiological levels of E2 and P4 and changes (depending on the degree of stimulation) in the E2:P4 ratio, triggers abnormal expression of uterine AQP during pregnancy, and this is associated with implantation failure. These findings lead us to recognize that abnormal expression would also occur under any pathological state (such as endometriosis) that is associated with changes in the normal E2:P4 ratio. Thus, infertility among these patients might in part be linked to abnormal uterine AQP expression., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The steroid hormones are instrumental for the growth of mammary epithelial cells. Our previous study indicates that hormones regulate the expression of Rspondin-1 (Rspo1). Yet, the regulatory mechanism remains unknown. In the current study, we identify Amphiregulin (Areg) as a novel upstream regulator of Rspo1 expression mediating the hormonal influence. In response to hormonal signaling, Areg emanating from estrogen receptor (ER)-positive luminal cells, induce the expression of Rspo1 in ER-negative luminal cells. The paracrine action of Areg on Rspo1 expression is dependent on Egfr. Our data reveal a novel Estrogen-Areg-Rspo1 regulatory axis in the mammary gland, providing new evidence for the orchestrated action of systemic hormones and local growth factors., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Oviducts play an important role in the reproductive process, such as in gamete transport, fertilization, and early embryonic development. However, the regulation of oviductal function during luteal formation phase (3-5 days post-ovulation), which is a crucial phase for early embryonic development, remains poorly understood. This study investigated the roles of oviductal estradiol-17β (E2) and progesterone (P4) concentrations on bovine oviductal functions in the luteal formation phase using RT-qPCR for some genes of oviductal epithelial cells. Bovine oviducts ipsilateral to the corpus luteum (CL) in the luteal formation phase were collected from a slaughterhouse. The concentration of oviductal E2 was positively correlated with the mRNA expressions of nuclear P4 receptor (PGR) and protein disulfide isomerase family A member 4 (PDIA4), which is related to protein secretion, in the ampulla and with estrogen receptor α (ESR1) mRNA expression in the isthmus. In contrast, the concentration of oviductal P4 was not correlated with oviductal mRNA expressions in either regions. Furthermore, for the candidate factor related to the oviductal E2 concentration, the CL parameters (CL size and tissue P4 concentration), first-wave dominant follicle (W1DF) parameters (follicle size and intrafollicular E2 concentration), and W1DF location (ipsilateral or contralateral to CL) did not influence the oviductal E2 concentration. In conclusion, our results suggest that the local oviductal E2 is a potential oviductal function regulator during the luteal formation phase.

Endometriosis is an oestrogen-dependent disease, and epithelial-mesenchymal transition (EMT) is involved in the process of endometriosis. Whether oestrogen could induce EMT in endometriosis remains largely unknown. Here, we reported that up-regulated expression of EMT markers in ovarian chocolate cyst is accompanied by high expression 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1), and exposure of primary human endometrial epithelial cells to oestradiol conditions could promote EMT occurrence and activate both β-catenin and Snail signalling. Furthermore, we found nuclear β-catenin and Snail expression was closely linked in ovarian endometriosis, and β-catenin knockdown abrogated oestrogen-induced Snail mediated EMT in vitro. This is due to that β-catenin/ TCF-3 could bind to Snail promoter and activate its transcription. These results suggested that β-catenin signalling functions as the Snail activator and plays a critical role in oestradiol-induced EMT in endometriosis., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Estradiol acutely facilitates sex differences in striatum-dependent behaviors. However, little is understood regarding the underlying mechanism. In striatal regions in adult rodents, estrogen receptors feature exclusively extranuclear expression, suggesting that estradiol rapidly modulates striatal neurons. We tested the hypothesis that estradiol rapidly modulates excitatory synapse properties onto medium spiny neurons (MSNs) of two striatal regions, the nucleus accumbens core and caudate-putamen in adult female and male rats. We predicted there would be sex-specific differences in pre- and postsynaptic locus and sensitivity. We further analyzed whether MSN intrinsic properties are predictive of estrogen sensitivity. Estradiol exhibited sex-specific acute effects in the nucleus accumbens core: miniature excitatory postsynaptic current (mEPSC) frequency robustly decreased in response to estradiol in female MSNs, and mEPSC amplitude moderately increased in response to estradiol in both male and female MSNs. This increase in mEPSC amplitude is associated with MSNs featuring increased intrinsic excitability. No MSN intrinsic electrical property associated with changes in mEPSC frequency. Estradiol did not acutely modulate mEPSC properties in the caudate-putamen of either sex. This is the first demonstration of acute estradiol action on MSN excitatory synapse function. This demonstration of sex and striatal region-specific acute estradiol neuromodulation revises our understanding of sex hormone action on striatal physiology and resulting behaviors. NEW & NOTEWORTHY This study is the first to demonstrate rapid estradiol neuromodulation of glutamatergic signaling on medium spiny neurons (MSNs), the major output neuron of the striatum. These findings emphasize that sex is a significant biological variable both in MSN sensitivity to estradiol and in pre- and postsynaptic mechanisms of glutamatergic signaling. MSNs in different regions exhibit diverse responses to estradiol. Sex- and region-specific estradiol-induced changes to excitatory signaling on MSNs explain sex differences partially underlying striatum-mediated behaviors and diseases.

Navigating a changing environment requires associating stimuli and actions with their likely outcomes and modifying these associations when they change. These processes involve the orbitofrontal cortex (OFC). Although some molecular mediators have been identified, developmental factors are virtually unknown. We hypothesized that the cell adhesion factor β1-integrin is essential to OFC function, anticipating developmental windows during which β1-integrins might be more influential than others. We discovered that OFC-selective β1-integrin silencing before adolescence, but not later, impaired the ability of mice to extinguish conditioned fear and select actions based on their likely outcomes. Early-life knock-down also reduced the densities of dendritic spines, the primary sites of excitatory plasticity in the brain, and weakened sensitivity to cortical inputs. Notwithstanding these defects in male mice, females were resilient to OFC (but not hippocampal) β1-integrin loss. Existing literature suggests that resilience may be explained by estradiol-mediated transactivation of β1-integrins and tropomyosin receptor kinase B (trkB). Accordingly, we discovered that a trkB agonist administered during adolescence corrected reward-related decision making in β1-integrin-deficient males. In sum, developmental β1-integrins are indispensable for OFC function later in life. SIGNIFICANCE STATEMENT The orbitofrontal cortex (OFC) is a subregion of the frontal cortex that allows organisms to link behaviors and stimuli with anticipated outcomes, and to make predictions about the consequences of one's behavior. Aspects of OFC development are particularly prolonged, extending well into adolescence, likely optimizing organisms' abilities to prospectively calculate the consequences of their actions and select behaviors appropriately; these decision making strategies improve as young individuals mature into adulthood. Molecular factors are not, however, well understood. Our experiments reveal that a cell adhesion protein termed "β1-integrin" is necessary for OFC neuronal maturation and function. Importantly, β1-integrins operate during a critical period equivalent to early adolescence in humans to optimize the ability of organisms to update expectancies later in life., (Copyright © 2019 the authors.)

Highlighted Research Paper: Chemogenetic Suppression of Medial Prefrontal-Dorsal Hippocampal Interactions Prevents Estrogenic Enhancement of Memory Consolidation in Female Mice by, Jennifer J. Tuscher, Lisa R. Taxier, Jayson C. Schalk, Jacqueline M. Haertel, and Karyn M. Frick.

Importance: Changes in estradiol during aging are associated with increased dementia risk. It remains unclear how estradiol supports cognitive health and whether risk factors, such as midlife obesity, are exacerbated by estrogen loss., Objectives: To assess whether visceral adipose tissue (VAT) moderates the association between age and brain network structure and to investigate whether estradiol moderates the association between VAT and brain network structure., Design, Setting, and Participants: Cross-sectional study of data from 974 cognitively healthy adults in Germany who participated in the Health Study of the Leipzig Research Centre for Civilization Diseases, a previously described population-based cohort study. Two moderation analyses were performed, including VAT as the moderator variable between age and brain network structure and estradiol as the moderator variable between VAT and brain network structure. The study was conducted from August 1, 2011, to November 23, 2014. Analyses were conducted from August 2017 to September 2018., Exposures: Serum estradiol levels from fasting blood and visceral adipose tissue volume from T1-weighted magnetic resonance imaging (MRI)., Main Outcomes and Measures: Brain network covariance (individual loading on structural network derived from T1-weighted MRI) and memory performance (composite score from the Consortium to Establish a Registry for Alzheimer Disease [CERAD] verbal episodic memory test on learning [score range, 0-30], recall [score range, 0-10], and recognition [score range, 0-20])., Results: Final analyses included data from 473 women (mean [SD] age, 50.10 [15.63] years) and 501 men (mean [SD] age, 51.24 [15.67] years). Visceral adipose tissue was associated with an exacerbation of the negative association of aging with network covariance for women (interaction term β = -0.02; 95% bias-corrected bootstrap CI, -0.03 to -0.01; P = .001) and men (interaction term β = -0.02; 95% bias-corrected bootstrap CI, -0.03 to -0.01; P < .001). Estradiol level was associated with a reduction in the negative association of VAT with network covariance in women (interaction term β = 0.63; 95% bias-corrected bootstrap CI, 0.14-1.12; P = .01), with no significant association in men. In the female midlife subgroup (age range, 35-55 years, when menopause transition occurs), low estradiol levels were associated with lower memory network covariance (Cohen d = 0.61; t80 = 2.76; P = .007) and worse memory performance (Cohen d = 0.63; t76 = 2.76; P = .007)., Conclusions and Relevance: This study reports a novel association between VAT, estradiol, and structural brain networks as a potential mechanism underlying cognitive decline in women. These findings appear to highlight the need for sex-specific strategies, including VAT and hormonal screening during midlife, to support healthy cognitive aging.

Social hierarchies emerge when animals compete for access to resources such as food, mates or physical space. Wild and laboratory male mice have been shown to develop linear hierarchies, however, less is known regarding whether female mice have sufficient intrasexual competition to establish significant social dominance relationships. In this study, we examined whether groups of outbred CD-1 virgin female mice housed in a large vivaria formed social hierarchies. We show that females use fighting, chasing and mounting behaviors to rapidly establish highly directionally consistent social relationships. Notably, these female hierarchies are less linear, steep and despotic compared to male hierarchies. Female estrus state was not found to have a significant effect on aggressive behavior, though dominant females had elongated estrus cycles (due to increased time in estrus) compared to subordinate females. Plasma estradiol levels were equivalent between dominant and subordinate females. Subordinate females had significantly higher levels of basal corticosterone compared to dominant females. Analyses of gene expression in the ventromedial hypothalamus indicated that subordinate females have elevated ERα, ERβ and OTR mRNA compared to dominant females. This study provides a methodological framework for the study of the neuroendocrine basis of female social aggression and dominance in laboratory mice.

In male songbirds, the motivation to sing is largely regulated by testosterone (T) action in the medial preoptic area, whereas T acts on song control nuclei to modulate aspects of song quality. Stereotaxic implantation of T in the medial preoptic nucleus (POM) of castrated male canaries activates a high rate of singing activity, albeit with a longer latency than after systemic T treatment. Systemic T also increases the occurrence of male-like song in female canaries. We hypothesized that this effect is also mediated by T action in the POM. Females were stereotaxically implanted with either T or with 17β-estradiol (E2) targeted at the POM and their singing activity was recorded daily during 2 h for 28 d until brains were collected for histological analyses. Following identification of implant localizations, three groups of subjects were constituted that had either T or E2 implanted in the POM or had an implant that had missed the POM (Out). T and E2 in POM significantly increased the number of songs produced and the percentage of time spent singing as compared with the Out group. The songs produced were in general of a short duration and of poor quality. This effect was not associated with an increase in HVC volume as observed in males, but T in POM enhanced neurogenesis in HVC, as reflected by an increased density of doublecortin-immunoreactive (DCX-ir) multipolar neurons. These data indicate that, in female canaries, T acting in the POM plays a significant role in hormone-induced increases in the motivation to sing., (Copyright © 2019 Vandries et al.)

Androgens and estrogens are known to be critical regulators of mammalian physiology and development. While these two classes of steroids share similar structures (in general, estrogens are derived from androgens via the enzyme aromatase), they subserve markedly different functions via their specific receptors. In the past, estrogens such as estradiol were thought to be most important in the regulation of female biology, while androgens such as testosterone and dihydrotestosterone were believed to primarily modulate development and physiology in males. However, the emergence of patients with deficiencies in androgen or estrogen hormone synthesis or actions, as well as the development of animal models that specifically target androgen- or estrogen-mediated signaling pathways, have revealed that estrogens and androgens regulate critical biological and pathological processes in both males and females. In fact, the concept of "male" and "female" hormones is an oversimplification of a complex developmental and biological network of steroid actions that directly impacts many organs. In this Review, we will discuss important roles of estrogens in males and androgens in females.

Backgrounds/aim: Male and female hearts have many structural and functional differences. Here, we investigated the role of estrogen (E2) in the mechanisms of sex differences in contraction through the cAMP-L-type Ca 2+ channel pathway in adult mice left ventricular (LV) apical myocytes at basal and stress state., Methods: Isolated LV apical myocytes from male, female (Sham) and ovariectomised mice (OVX) were used to investigate contractility, Ca 2+ transients and L-type Ca 2+ channel (LTCC) function. The levels of β 2 AR, intracellular cAMP, phosphodiesterase (PDE 3 and PDE 4), RyR2, PLB, SLN, and SERCA2a were compared among the experimental groups., Results: We found that (1) intracellular cAMP, I CaL density, contraction and Ca 2+ transient amplitudes were larger in Sham and OVX + E2 myocytes compared to male and OVX. (2) The mRNA expression of PDE 3 and 4 were lower in Sham and OVX + E2 groups compared with male and OVX groups. Treatment of myocytes with IBMX (100 μM) increased contraction and Ca 2+ transient amplitude in both sexes and canceled differences between them. (3) β 2 AR-mediated stress decreased cAMP concentration and peak contraction and Ca 2+ transient amplitude only in male and OVX groups but not in Sham or OVX + E2 groups suggesting a cardioprotective role of E2 in female mice. (4) Pretreatment of OVX myocytes with GPR30 antagonist G15 (100 nM) abolished the effects of E2, but ERα and ERβ antagonist ICI 182,780 (1 μM) did not. Moreover, activation of GPR30 with G1 (100 nM) replicated the effects of E2 on cAMP, contraction and Ca 2+ transient amplitudes suggesting that the acute effects of E2 were mediated by GPR30 via non-genomic signaling. (5) mRNA expression of RyR2 was higher in myocytes from Sham than those of male while PLB and SLN were higher in male than Sham but no sex differences were observed in the mRNA of SERCA2a., Conclusion: Collectively, these results demonstrate that E2 modulates the expression of genes related to the cAMP-LTCC pathway and contributes to sex differences in cardiac contraction and responses to stress. We also show that estrogen confers cardioprotection against cardiac stress by non-genomic acute signaling via GPR30.

17β-estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but its precise functions in the brain are unclear. Here, we used a forebrain-neuron-specific aromatase knock-out (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain of mice and thereby elucidate its functions. FBN-ARO-KO mice showed a 70-80% decrease in aromatase and forebrain E2 levels compared with FLOX controls. Male and female FBN-ARO-KO mice exhibited significant deficits in forebrain spine and synaptic density, as well as hippocampal-dependent spatial reference memory, recognition memory, and contextual fear memory, but had normal locomotor function and anxiety levels. Reinstating forebrain E2 levels via exogenous in vivo E2 administration was able to rescue both the molecular and behavioral defects in FBN-ARO-KO mice. Furthermore, in vitro studies using FBN-ARO-KO hippocampal slices revealed that, whereas induction of long-term potentiation (LTP) was normal, the amplitude was significantly decreased. Intriguingly, the LTP defect could be fully rescued by acute E2 treatment in vitro Mechanistic studies revealed that FBN-ARO-KO mice had compromised rapid kinase (AKT, ERK) and CREB-BDNF signaling in the hippocampus and cerebral cortex. In addition, acute E2 rescue of LTP in hippocampal FBN-ARO-KO slices could be blocked by administration of a MEK/ERK inhibitor, further suggesting a key role for rapid ERK signaling in neuronal E2 effects. In conclusion, the findings provide evidence of a critical role for neuron-derived E2 in regulating synaptic plasticity and cognitive function in the male and female brain. SIGNIFICANCE STATEMENT The steroid hormone 17β-estradiol (E2) is well known to be produced in the ovaries in females. Intriguingly, forebrain neurons also express aromatase, the E2 biosynthetic enzyme, but the precise functions of neuron-derived E2 is unclear. Using a novel forebrain-neuron-specific aromatase knock-out mouse model to deplete neuron-derived E2, the current study provides direct genetic evidence of a critical role for neuron-derived E2 in the regulation of rapid AKT-ERK and CREB-BDNF signaling in the mouse forebrain and demonstrates that neuron-derived E2 is essential for normal expression of LTP, synaptic plasticity, and cognitive function in both the male and female brain. These findings suggest that neuron-derived E2 functions as a novel neuromodulator in the forebrain to control synaptic plasticity and cognitive function., (Copyright © 2019 Lu, Sareddy et al.)

Sex hormones, such as testosterone, progesterone, and 17β-estradiol, control various physiological functions. This review focuses on the sex hormonal regulation of K + channels and the effects of such regulation on electrophysiological and contractile functions of muscles. In the cardiac tissue, testosterone and progesterone shorten action potential, and estrogen lengthens QT interval, a marker of increased risk of ventricular tachyarrhythmias. We have shown that testosterone and progesterone in physiological concentration activate KCNQ1 channels via membrane-delimited sex hormone receptor/eNOS pathways to shorten the action potential duration. Mitochondrial K + channels are also involved in the protection of cardiac muscle. Testosterone and 17β-estradiol directly activate mitochondrial inner membrane K + channels (Ca 2+ activated K + channel (K Ca channel) and ATP-sensitive K + channel (K ATP channel)) that are involved in ischemic preconditioning and cardiac protection. During pregnancy, uterine blood flow increases to support fetal growth and development. It has been reported that 17β-estradiol directly activates large-conductance Ca 2+ -activated K + channel (BK Ca channel) attenuating arterial contraction. Furthermore, 17β-estradiol increases expression of BK Ca channel β1 subunit which enhances BK Ca channel activity by DNA demethylation. These findings are useful for understanding the mechanisms of sex or generation-dependent differences in the physiological and pathological functions of muscles, and the mechanisms of drug actions., (Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Although the 2017 Endocrine Society Guidelines for gender dysphoria stipulated that cross-sex hormone therapy (CHT) achieve gonadal steroid levels equivalent to those of a cisperson of the chosen sex, for transgender women (male-to-female gender dysphoria), current gonadal therapy is usually estradiol. Accumulated evidence indicates that normally ovulatory menstrual cycles are necessary for ciswomen's current fertility, as well as for later-life bone and cardiovascular health and the prevention of breast and endometrial cancers., Evidence Acquisition: Extensive past clinical experience with transgender women's CHT using estradiol/estrogen combined with progesterone/medroxyprogesterone and pioneering the addition of spironolactone. Comprehensive progesterone physiology research plus a brief review of transgender women's literature to assess current therapy and clinical outcomes, including morbidity and mortality., Purpose: To emphasize that both ovarian hormones, progesterone as well as estradiol, are theoretically and clinically important for optimal transgender women's CHT., Evidence Synthesis: It is important to add progesterone to estradiol and an antiandrogen in transgender women's CHT. Progesterone may add the following: (i) more rapid feminization, (ii) decreased endogenous testosterone production, (iii) optimal breast maturation to Tanner stages 4/5, (iv) increased bone formation, (v) improved sleep and vasomotor symptom control, and (vi) cardiovascular health benefits., Conclusions: Evidence has accrued that normal progesterone (and ovulation), as well as physiological estradiol levels, is necessary during ciswomen's premenopausal menstrual cycles for current fertility and long-term health; transgender women deserve progesterone therapy and similar potential physiological benefits., (Copyright © 2019 Endocrine Society.)

Central output of gonadotropin-releasing hormone (GnRH) neurons controls fertility and is sculpted by sex-steroid feedback. A switch of estradiol action from negative to positive feedback initiates a surge of GnRH release, culminating in ovulation. In ovariectomized mice bearing constant-release estradiol implants (OVX+E), GnRH neuron firing is suppressed in the morning (AM) by negative feedback and activated in the afternoon (PM) by positive feedback; no time-of-day-dependent changes occur in OVX mice. In this daily surge model, GnRH neuron intrinsic properties are shifted to favor increased firing during positive feedback. It is unclear whether this shift and the observed concomitant increase in GABAergic transmission, which typically excites GnRH neurons, are independently sufficient for increasing GnRH neuron firing rate during positive feedback or whether both are needed. To test this, we used dynamic clamp to inject selected previously recorded trains of GABAergic postsynaptic conductances (PSgs) collected during the different feedback states of the daily surge model into GnRH neurons from OVX, OVX+E AM, and OVX+E PM mice. PSg trains mimicking positive feedback initiated more action potentials in cells from OVX+E PM mice than negative feedback or OVX (open feedback loop) trains in all three animal models, but the positive-feedback train was most effective when applied to cells during positive feedback. In silico studies of model GnRH neurons in which >1000 PSg trains were tested exhibited the same results. These observations support the hypothesis that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during positive feedback. SIGNIFICANCE STATEMENT Infertility affects 15%-20% of couples; failure to ovulate is a common cause. Understanding how the brain controls ovulation is critical for new developments in both infertility treatment and contraception. Ovarian estradiol alters both the intrinsic properties of gonadotropin-releasing hormone (GnRH) neurons and synaptic inputs to these cells coincident with production of sustained GnRH release that ultimately triggers ovulation. We demonstrate here using dynamic clamp and mathematical modeling that estradiol-induced shifts in synaptic transmission alone can increase firing output, but that the intrinsic properties of GnRH neurons during positive feedback further poise these cells for increased response to higher frequency synaptic transmission. These data suggest that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during the preovulatory GnRH surge., (Copyright © 2019 the authors 0270-6474/19/392092-11$15.00/0.)

Post-traumatic stress disorder (PTSD) is more prevalent in women than men, yet much remains to be determined regarding the mechanism underlying this sex difference. Clinical and preclinical studies have shown that low estradiol levels during extinction of fear conditioning in rodents (i.e., cue exposure therapy in humans) leads to poor extinction consolidation and increased fear during extinction recall. The renin-angiotensin system (RAS) is also associated with stress-related pathologies, and RAS antagonists can enhance extinction consolidation in males. However, less is known about how estradiol and the RAS converge to alter fear extinction consolidation in females. Since estradiol downregulates the RAS, we determined the role of surgically (via ovariectomy [OVX]) and pharmacologically (via the hormonal contraceptive [HC], levonorgestrel) clamping estradiol at low levels in female rats on fear-related behavior, serum estradiol and angiotensin II (Ang II) levels, and angiotensin II type I receptor (AT1R) binding in the brain. We then tested whether the AT1R antagonist losartan would alter fear-related behavior in an estradiol-dependent manner. We found that both OVX and HC treatment produced extinction consolidation deficits relative to intact female rats in proestrus (when estradiol levels are high), and that losartan treatment mitigated these deficits and reduced freezing. OVX, but not HC, altered AT1R ligand binding, though HC reduced estradiol and increased Ang II levels in plasma. These findings have significant clinical implications, indicating that administration of an AT1R antagonist, especially if estradiol levels are low, prior to an exposure therapy session may improve treatment outcomes in females.

Background: Melatonin is an amine hormone that plays an important role in regulating mammalian reproduction. This study aimed to investigate the expression pattern of melatonin synthesis enzymes AANAT and HIOMT and melatonin receptors MT1 and MT2 in sheep cumulus-oocyte complexes (COCs) as well as the change of melatonin level in follicular fluid (FF) during antral follicle development. In this research, we also study the effect of β-estradiol (E2) on MT1 and MT2 expression as well as melatonin synthesis in COCs so as to lay the foundation for further exploration of the regulation mechanism of melatonin synthesis in the ovary., Methods: COCs and FF were collected from different size (large follicles (diameter ≥ 5 mm), medium follicles (diameter 2-5 mm), and small follicles (diameter ≤ 2 mm)) of antral follicles in sheep ovaries. To assess whether E2 regulates melatonin synthase and its receptors expression in sheep COCs and whether it is mediated through estrogen receptor (ER) pathway. The collected COCs were cultured in vitro for 24 h and then treat with 1 μM E2 and/or 1 μM ICI182780 (non-selective ER antagonist). The expression of AANAT, HIOMT, MT1 and MT2 mRNA and protein were determined by qRT-PCR and western blot. The melatonin level was determined by ELISA., Results: The expression of AANAT, HIOMT, MT1 and MT2 were significantly higher expression in the COCs of small follicles than in those of large follicles (P < 0.05). However, the melatonin level was significantly higher in large follicle FF than in small follicle FF (P < 0.05). Further, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production were decreased by E2 treatment (P < 0.05), but when ICI182780 was added, the expression of AANAT, HIOMT, MT1, and MT2 and melatonin production recovered (P < 0.05)., Conclusions: We suggest that sheep COCs can synthesize melatonin, but this ability is decreased with increasing follicle diameter. Furthermore, E2 play an important role in regulated the expression of MT1 and MT2 as well as melatonin synthesis in sheep COCs through the ER pathway.

Background: In women, changes in estrogen levels may increase the incidence and/or symptomatology of depression and affect the response to antidepressant treatments. Estrogen therapy in females may provide some mood benefits as a single treatment or might augment clinical response to antidepressants that inhibit serotonin reuptake., Objective: We analyzed the mechanisms of estradiol action involved in the regulation of gene expression that modulates serotonin neurotransmission implicated in depression., Method: Publications were identified by a literature search on PubMed., Results: The participation of estradiol in depression may include regulation of the expression of tryptophan hydroxylase-2, monoamine oxidase A and B, serotonin transporter and serotonin-1A receptor. This effect is mediated by estradiol binding to intracellular estrogen receptor that interacts with estrogen response elements in the promoter sequences of tryptophan hydroxylase-2, serotonin transporter and monoamine oxidase-B. In addition to directly binding deoxyribonucleic acid, estrogen receptor can tether to other transcription factors, including activator protein 1, specificity protein 1, CCAAT/enhancer binding protein β and nuclear factor kappa B to regulate gene promoters that lack estrogen response elements, such as monoamine oxidase-A and serotonin 1A receptor., Conclusion: Estradiol increases tryptophan hydroxylase-2 and serotonin transporter expression and decreases the expression of serotonin 1A receptor and monoamine oxidase A and B through the interaction with its intracellular receptors. The understanding of molecular mechanisms of estradiol regulation on the protein expression that modulates serotonin neurotransmission will be helpful for the development of new and more effective treatment for women with depression., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Endogenous ovarian hormones as well as exogenous oestradiol and progesterone play an important role in cognitive processing. Specifically, these hormones play a role in different aspects of memory, both in terms of storage capacity and temporal duration of the mnemonic track. These hormones also have various effects on different types of memory (i.e., verbal, visuo-spatial, prospective). This study investigated the effects of hormones on topographic memory, a type of memory specifically needed to recall a pathway and to acquire spatial information about locations, distances, and directions., Methods: We compared 25 naturally cycling women (NCW) in two different cycling phases, the early follicular phase (4th - 5th days) and the mid-luteal phase (20th-21st days), with 26 women taking oral contraceptives (OC) tested in the active pill phase (20th to 21st day of OC cycle) and the inactive pill phase (2nd to 4th day of OC cycle). Both groups performed the Walking Corsi Test to assess topographic memory in their respective cycling phases. Women were instructed to learn an eight-step sequence path and recall the path five minutes later., Results: We found that the two groups differed in terms of learning the 8-step sequence path; OC users were always better (4-5 days vs. 20-21 days) than NCW. No differences emerged in the delayed recall of the same path., Conclusions: As already observed in other memory domains (i.e., verbal memory, emotional memory), OC users showed an advantage in terms of topographic learning. Our results might be explained by hormonal mechanisms and may suggest the future application of OC in women with topographic disorders or visuo-spatial difficulties.

Purpose: This study aimed to determine the effects of 5 months of ovarian hormone suppression in premenopausal women on objectively measured physical activity (PA)., Methods: Participants (age, 35 ± 8 yr; body mass index, 27 ± 6 kg·m) received monthly intramuscular injections of gonadotropin-releasing hormone agonist (GnRHAG) therapy, which suppresses pituitary gonadotropins and results in suppression of ovarian sex hormones. Women were randomized to receive concurrent transdermal E2 (GnRHAG + E2; n = 30) or placebo (GnRHAG + PL, n = 31). PA was assessed for 1 wk before and during each month of the 5-month intervention using a hip-worn accelerometer (Actical, Mini Mitter Co., Inc., Bend, OR). Estimates of time spent in sedentary, light, and moderate-to-vigorous PA (MVPA) were derived using a previously published equation. Subsets of participants in each group were also randomized to a supervised progressive resistance exercise training program., Results: Total MVPA tended toward being higher (P = 0.08) in the GnRHAG + E2 group at month 4. There were no significant effects of intervention or time in sedentary or light PA. In the subset of women who did not participate in structured exercise training for which Actical data were obtained (n = 16 in each group), total MVPA was higher at month 4 (P = 0.01)., Conclusions: PA levels seem to be maintained at a higher level in women undergoing pharmacological suppression of ovarian function with E2 add-back when compared with women treated with placebo. These data provide proof-of-concept data that E2 contributes to the regulation of PA in humans. However, given the exploratory nature of this study, future confirmatory investigations will be necessary.

The biological mechanisms regulating physical activity patterns appear to be linked to the sex hormones. Elucidation of these regulatory mechanisms may enhance individual physical activity patterns producing positive gains in health., Purpose: The purpose of this study was to evaluate the prolonged effects of estrogen on wheel running distance, duration, and speed in orchidectomized mice., Methods: The physical activity patterns of 9-wk-old C57BL/6j male mice (n = 28) were observed. Wheel running distance, duration, and speed were assessed under physiological conditions for 7 d. Next, physical activity patterns were evaluated after bilateral orchidectomy (n = 14) or sham orchidectomy (n = 14) for an additional 7 d. Orchidectomized mice were provided estrogen containing capsules for three additional weeks; control mice were provided estrogen-free capsules. Wheel running distance, duration, and speed were analyzed by three two-way (treatment group-phase of study) analysis of variance tests., Results: Wheel running speed was unaffected by sex hormone status. Distance (mean ± SD = 6.74 ± 2.13 km at baseline) decreased significantly after orchidectomy (2.27 ± 1.55 km) and remained low after initial estrogen treatment (3.04 ± 1.05 km). Prolonged estrogen exposure sustained a significant elevation of daily distance (4.47 ± 1.87 km). Prolonged estrogen exposure recovered and significantly sustained wheel running duration (baseline, 248 ± 60 min; postorchidectomy, 102 ± 53 min; prolonged exposure, 170 ± 63 min)., Conclusions: Wheel running behavior was reduced significantly after orchidectomy and remained low after initial treatment with estrogens, but recovered to near control levels after 2 wk of exposure to estrogens. The estrogenic mechanism regulating wheel running behavior in male mice appears to induce an extensive but slow acting biological mechanism. Understanding the biological drive behind this mechanism may aid in developing useful therapeutic strategies to combat health issues related to physical inactivity.

Context: Supraphysiological estradiol exposure after ovarian stimulation may disrupt embryo implantation after fresh embryo transfer. Women with polycystic ovary syndrome (PCOS), who usually overrespond to ovarian stimulation, have a better live birth rate after frozen embryo transfer (FET) than after fresh embryo transfer; however, ovulatory women do not., Objective: To evaluate whether the discrepancy in live birth rate after fresh embryo transfer vs FET between these two populations is due to the variation in ovarian response (i.e., peak estradiol level or oocyte number)., Design, Setting, Patients, Intervention(s), and Main Outcome Measure(s): This was a secondary analysis of data from two multicenter randomized trials with similar study designs. A total of 1508 women with PCOS and 2157 ovulatory women were randomly assigned to undergo fresh or FET. The primary outcome was live birth., Results: Compared with fresh embryo transfer, FET resulted in a higher live birth rate (51.9% vs 40.7%; OR, 1.57; 95% CI, 1.22 to 2.03) in PCOS women with peak estradiol level >3000pg/mL but not in those with estradiol level ≤3000 pg/mL. In women with PCOS who have ≥16 oocytes, FET yielded a higher live birth rate (54.8% vs 42.1%; OR, 1.67; 95% CI, 1.20 to 2.31), but this was not seen in those with <16 oocytes. In ovulatory women, pregnancy outcomes were similar after fresh embryo transfer and FET in all subgroups., Conclusions: Supraphysiological estradiol after ovarian stimulation may adversely affect pregnancy outcomes in women with PCOS but not in ovulatory women.